The Molecular Mechanism Of RNA-DNA Hybrids In Regulating Meiotic Recombination

Meiosis is a specialized cell division to generate mature germ cells that occurs in sexually reproducing organisms.A central feature of meiosis is the generation and repair of DNA double-strand break(DSB).During meiosis I,a large number of programmed DSBs mediated by Spol 1 complexes are produced.Cells use homologs as templates to repair DSBs,and generate crossover(CO)eventually which result in the correct separation of homologous chromosomes and increase genetic diversity.RNA transcripts can anneal with template DNA strands to form RNA-DNA hybrids.RNA-DNA hybrids play an important role in many physiological processes such as DNA replication,transcription and gene expression.However,when its level is abnormal,it will lead to DNA damage and genome instability,so there are a variety of factors regulating the level of RNA-DNA hybrids in cells.RNase H nuclease family containing RNase H1 and RNase H2,which can specifically recognize and cut the RNA chain in RNA-DNA hybrids,elimination of the RNA-DNA hybrids that already formed.As well as the THO complex,facilitates rapid transport of mRNA out of the nucleus,preventing the production of RNA-DNA hybrids.Recent studies have shown that RNADNA hybrids have vital function in mitotic homologous recombination,but their role in meiosis is still poorly understood.Saccharomyces cerevisiae was used as genetic material in this study to construct mutant strains with excessive accumulation and excessive reduction of RNA-DNA hybrids.Immunofluorescence、DRIP、DNA 1D/2D-Southern and whole-genome resequencing were used to explore the role of RNA-DNA hybrids in the regulation of meiotic homologous recombination was explored.The main results are as follows:We deleted the subunits of RNase H1,RNase H2 and THO complexes,and constructed a series of mutant strains with RNA-DNA hybrids accumulation,rnh1Δ、rnh201Δ、hpr1Δ、rnh1Δrnh20Δ、rnh1Δrnh204Δhpr1Δ,to explore the role of RNADNA hybrids in meiosis.We used S9.6 antibody(an antibody that can specifically detect RNA-DNA hybrids)to exam the levels of RNA-DNA hybrids in the wild type and mutants.RNA-DNA hybrids were barely detected by immunostaining in WT meiosis,suggesting that they are effectively prevented,or timely removed,and thus maintained at a low level.But a large amount of RNA-DNA hybrids signal were observed in the rnh1Δrnh201Δ and rnhlArnh201Δhpr1Δ mutants.To explore the effects of accumulated RNA-DNA hybrids on meiosis,the meiotic time-course analysis、sporulation efficiency、spore viability was performed.Compared to WT,the meiotic nuclear division was delayed,sporulation efficiency and spore viability were both decreased.Therefore,the accumulation of RNA-DNA hybrids causes multiple meiotic defects.To explore the roles of RNA-DNA hybrids in meiotic recombination,coimmunostaining of RNA-DNA hybrids and recombination factor RPA was performed in surface spread meiotic nuclei.Compared to WT,the repair of DSBs in rnh1Δrnh201Δ hpr1Δ mutant was delayed by 1h,reflecting the defective of DSBs repair.The results of co-immunostaining showed that～75%RNA-DNA hybrid foci colocalized with RPA foci suggest that RNA-DNA hybrids preferentially form at meiotic recombination sites,and raises the possibility that the formation of RNA-DNA hybrids depends on meiotic DSBs.Hence,we introduced a catalytic inactive spoll allele(spo11Y135F)into the rnhlΔrnh20Δhpr1Δ triple mutant to eliminate meiotic DSB formation.The maximal number of RNA-DNA hybrid foci in meiotic cells dramatically decreased to～20%of those in rnh1Δrnh201Δhpr1Δ,indicating that～80%RNA-DNA hybrids were meiotic DSB-dependent in the triple mutant.The result suggest that the formation of most meiotic RNA-DNA hybrids depends on meiotic DSBs.Given that the formation of meiotic RNA-DNA hybrids depends on programmed DSBs and ssDNA ends of resected DSBs are good substrates for RNA binding,we wondered whether the formation of meiotic RNA-DNA hybrids requires the existence of ssDNA ends.Hence,we introduced rad50S,exo1Δ,or dmc1Δ,predicted to differently affect end resection,into rnhlΔrnh201Δhpr1Δ or rnh1Δrnh201Δ mutant.When the DSB ends remain unresected,RNA-DNA hybrids were not detected by immunostaining,and when the DSB ends are over-resected to give super long ssDNA,RNA-DNA hybrids were accumulated,indicated that the formation of meiotic RNADNA hybrids requires the existence of ssDNA ends.This hypothesis is further supported by the fact that RNA-DNA hybrids were enriched in the proximity of breaking sites at both HIS4LEU2 and BUD23 DSB hotspots,as assayed by DRIP-qPCR.Detailed analysis showed that RNA-DNA hybrids asymmetrically flanked DSB hotspots and the flank with more RNA-DNA hybrids has more transcripts.According this result,we wonder whether meiotic RNA-DNA hybrids are produced by in situ transcription or by transcripts reannealed with DNA templates.We specifically deplete Rad52 in the rnh1Δrnh201Δhpr1Δ strain during meiosis resulted in gradually decreased numbers of RNA-DNA hybrids during meiosis.Therefore,the abundant accumulation of RNA-DNA hybrids during meiosis depends on the level of Rad52 and most(if not all)meiotic RNA-DNA hybrids likely form in trans.Accumulated meiotic RNA-DNA hybrids on recombination sites delayed DSB repair,which finally resulted in multiple meiotic defects.To further investigate the effects of accumulated RNA-DNA hybrids on meiotic recombination,we examined the number of Zip3 foci,which specifically mark patterned COs at pachytene,in rnh1Δrnh201Δhpr1Δ mutant.The number of Zip3 foci was significantly decreased to～80%of Wild Type.Consistently,whole-genome re-sequencing of four-viable spores from a single tetrad showed that the total number of COs in the rnh1Δrnh201Δ mutant were decreased,too.To further dissect how accumulated RNA-DNA hybrids regulate meiotic recombination,a well-characterized DNA physical assay system at the HIS4LEU2 recombination hotspot was used to monitor meiotic DSBs,COs,NCOs,and DNA recombination intermediates,dHJs.Consistent with the cytological results,CO analyses showed that the level of COs decreased in rnh1Δrnh201ΔhprlΔ mutant,and the appearance of COs was delayed.And the ritro of IH-dHJs to IS-dHJs were decreased,reflecting the increased of sister bias,suggest the impair of meiotic homolog bias.RNA-DNA hybrids accumulated at DSB sites may compete with ssDNA binding proteins like Rad51 or Dmc1 to impair recombination.This idea was supported by our ChIP-qPCR results which showed significantly decreased enrichment of Rad51 and Dmc1 around DSB sites in rnh1Δrnh201ΔhprlΔ mutant.Although the numbers of Rad51 foci were not affected in the rnh1Δrnh201Δhpr1Δ mutant,Rad51 focus intensity in this mutant was weaker than that in WT.These results indicate that excessive RNADNA hybrids indeed compete with Rad51 to bind to DSB ends.These findings indicate that accumulated RNA-DNA hybrids compete with Rad51/Dmc1,impair meiotic homolog bias and,as a consequence,the levels of both COs and NCOs are reduced。To explore whether a reduced level of RNA-DNA hybrids affects homologous recombination,we used a copper-inducible CUP1 promoter to overexpress RNH1(pCUP1-RNH1)to precociously remove RNA-DNA hybrids in meiotic cells.Severe meiotic defects were observed when RNH1 were overexpressed,only 50%of cells can complete meiosis.And the level of meiosis recombination was reduced both cytologically and physically.These results indicated that RNA-DNA hybrids play an important role in meiosis,and excessive removal can lead to meiosis defectsds.In summary,the formation of meiotic RNA-DNA hybrids depends on meiotic DSBs and requires the existence of ssDNA ends,and abnormal accumulation or excessive removal of RNA-DNA hybrids will lead to reduced recombination levels and severe defects in meiosis.